A Novel Method for Single Molecule Visualization of Active Protein Synthesis in Cardiac Myocytes Reveals SERCA2a mRNA Translation is Sarcoplasmic Reticulum Ca2+ Load Dependent

Abstract

Protein abundance in cardiomyocytes is controlled at both transcription and translation levels. While changes in transcription in cardiac proteins such as the sarcoplasmic reticulum Ca2+ ATPAse (SERCA2a) in response to both physiological and pathological stimuli have been extensively studied, dynamic modulation of translation and the underlying mechanisms have received little attention. We have developed MR-PLISH (mRNA-rRNA Proximity Ligated in Situ Hybridization), a new imaging method for visualization of translation of endogenous specific mRNA transcripts in cardiomyocytes. This method is based on combined use of RNA in situ hybridization and the proximity ligation assay (PLA) technology and allows us to detect single mRNAs interacting with 18S ribosomal RNA (Rn18s). Using this approach, as well as RNAscope® for single molecule detection of mRNA, we examined the effects of depleting intra-SR Ca2+ with thapsigargin (TG) on transcription and translation of the mRNA for SERCA2a (Atp2a2) in cardiomyocytes. Under baseline conditions both total and 18s rRNA-hybridized Atp2a2 displayed a cell-wide distribution. Treatment with thapsigargin caused no significant changes in abundance or distribution of total Atp2a2 mRNA; however, it resulted in a significant increase in 18s rRNA-hybridized Atp2a2, consistent with an increased rate of active protein synthesis. These results suggest that intra-SR Ca2+ modulates localized translation of SERCA2a mRNA in cardiomyocytes. Thus, SERCA2A translation maybe subject to regulation via a local feedback loop to compensate for reduction in intra-SR Ca2+ levels.